Integral membrane protein complexes consisting of proteins and small molecules that act as cofactors are ubiquitous and vitally important for organisms. For example, cytochrome oxidase is a mitochondrial complex that catalyzes the delivery of electrons to oxygen to allow cellular respiration. Rhodopsin is found in the eyes of many animals, including humans, and consists of an opsin membrane protein and a Vitamin A retinal cofactor. In these systems, an exquisite balance of protein and cofactor is maintained. Disruptions in this balance are thought to be causative factors for diseases such as Alzheimer's and retinitis pigmentosa. The proposed project describes a multidisciplinary approach to characterize a novel mechanism for coordinated synthesis of proteins and cofactors for integral membrane complexes. We will take advantage of the simple bacteriorhodopsin (BR) protein-cofactor complex in the microbe Halobacterium salinarum to potentially provide insight into how coordination is achieved in more complex systems. BR functions as a light-driven proton pump in the halophilic archaeon H. salinarum, and allows the organism to generate usable cellular energy under conditions where low oxygen prevents aerobic respiration. BR is the simplest possible membrane protein complex consisting of a single retinal cofactor bound to a single protein, bacterioopsin (BO). Under low oxygen conditions, H. salinarum rapidly induces BR biosynthesis, and this induction necessarily requires increased production of both the protein, BO, and the cofactor, retinal. In the proposed work, we explore how H. salinarum proteins interact to coordinate the synthesis of BO and retinal. Our preliminary results suggest that this coordination occurs by a novel mechanism where BO, not bound by retinal, inhibits an alternate biochemical pathway to only allow precursor molecules to be used for retinal synthesis. To investigate this mechanism, we will first use genetic and biochemical approaches to characterize enzymes that catalyze the synthesis of the retinal cofactor and related molecules. We will then identify which retinal precursors or enzymes interact with BO. Lastly, we will structurally characterize the interaction between BO and these molecules. PUBLIC HEALTH RELEVANCE: Molecular complexes consisting of proteins and cofactors our critical to our everyday life. For example, rhodopsin is a complex consisting of a protein, opsin, and a Vitamin A molecule that function together to allow us to see. Cytochrome oxidase is a complex of several proteins and a heme molecule that come together to allow us to get energy from the food we eat. Unfortunately, these cofactors and proteins can occasionally get out of balance leading to diseases such as retinitis pigmentosa and Alzheimer's. The proposed research will study a similar molecular complex in a microbe in order to elucidate the mechanisms that keep proteins and their cofactors in the correct balance.